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The proteasomal system is involved in the turnover of damaged proteins and it is responsible for their degradation. Because of its role in oncogenesis, the inhibition of the proteasome system is a promising therapeutic target for neoplastic treatment. The accumulation and deleterious effects of toxic proteins are frequently induced by exposure to chemotherapeutic drugs and 20S proteasome inhibitors, such as bortezomib (BTZ) and carfilzomib (CFZ) have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of multiple myeloma (MM) and some other liquid tumours. Although the survival of MM patients has been improved by the introduction of both drugs-based therapies, these proteasome inhibitors have several limitations, including chemotherapy-induced peripheral neurotoxicity (CIPN). Since it is becoming increasingly clear that cytoskeletal integrity and mitochondrial function are intricately linked, we compared these targets after BTZ and CFZ treatments in vitro. Primary culture of dorsal root ganglion (DRG) sensory neurons isolated from adult mice were treated with BTZ 10 nM and CFZ 60 nM for 24 hours, and measurement of energy metabolism were investigated using XFe24 Seahorse Analyzer, while a morphological analysis of mitochondria was performed in silico using the toolset MiNA (Mitochondrial Network Analysis). Moreover, by immunoblotting we have evaluated drug-induced alterations of proteins involved in cytoskeleton, mitochondrial oxidative phosphorylation, and molecular motors transport. BTZ was shown to induce several cytoskeletal damage in terms of increased levels of delta2-tubulin, acetylated tubulin and MAP2 expressions compared to both CFZ-treated cells and untreated cells. Conversely, the evaluation of the bioenergetic mitochondrial metabolism revealed a reduction in basal and maximal respiration for both chemotherapeutic drugs. Similarly, both BTZ-and CFZ cultures showed a decrease in ATP production compared with the untreated cells.

Purpose: The study aims at investigating the impact of polymedication and aging in the prevalence of multiple drug-drug interactions (DDIs) on HCV patients treated with sofosbuvir/velpatasvir (SOF/VEL) or glecaprevir/pibrentasvir (GLE/PIB). Patients and Methods: This is a retrospective analysis based on administrative data covering around 6.9 million individuals. Patients treated with SOF/VEL or GLE/PIB over November 2017–March 2020 were included. Index date corresponded to SOF/VEL or GLE/PIB first prescription during such period; patients were followed up for treatment duration. Analyses were then focused on patients with ≥2 comedications at risk of multiple DDIs. The severity and the effect of multiple DDI were identified using the Liverpool University tool. Results: A total of 2057 patients with SOF/VEL and 2128 with GLE/PIB were selected. Mean age of SOF/VEL patients was 58.5 years, higher than GLE/PIB ones (52.5 years) (p < 0.001), and patients >50 years were more present in SOF/VEL vs GLE/PIB cohorts: 72% vs 58%, (p < 0.001). Most prescribed co-medications were cardiovascular, alimentary and nervous system drugs. Proportion of patients with ≥2 comedications was higher in SOF/VEL compared to GLE/PIB cohort (56.5% vs 32.3%, p < 0.001). Those at high-risk of multiple DDIs accounted for 11.6% (N = 135) of SOF/VEL and 19.6% (N = 135) of GLE/PIB (p < 0.001) patients with ≥2 comedications. Among them, the potential effect of DDI was a decrease of DAA serum levels (11% of SOF/VEL and GLE/PIB patients) and an increased concentration of comedication serum levels (14% of SOF/VEL and 42% of GLE/PIB patients). Conclusion: This real-world analysis provided a thorough characterization on the burden of polymedication regimens in HCV patients treated with SOF/VEL or GLE/PIB that expose such patients to an increased risk of DDIs. In our sample population, SOF/ VEL regimen was more frequently detected on elderly patients and on those with ≥2 comedications at risk

The causal mechanisms underlying chemotherapy-induced peripheral neuropathy (CIPN) are not yet fully understood, but primary afferent neurons have emerged as a vulnerable initiating pathophysiological target. Bortezomib (BTZ) is a proteasome inhibitor, which results in axonal degeneration, producing in patients a disabling painful peripheral neuropathy that undermine its therapeutic efficacy. In the current study, we examined whether treatment with BTZ for 24 hours would affect microtubule (MTs) stability as well as axonal transport and oxidative phosphorylation or mitochondrial bioenergetics in primary cultured dorsal root ganglion (DRG) sensory neurons. MT stability was indirectly measured by western blotting and quantitative immunofluorescence microscopy of delta2 tubulin and tubulin acetylation, while axonal mitochondrial trafficking was evaluated by time-lapse confocal microscopy and kymograph analysis. To evaluate the effects of BTZ on the generation and regulation of cellular bioenergetics, mitochondrial oxidative phosphorylation (OXPHOS) and fusion/fission balance were described via western blot analysis of key molecular markers, and real-time oxygen consumption rate (OCR), an indicator of mitochondrial respiration, was obtained by Seahorse bioanalyser. BTZ-treated sensory neurons induced an approximately 2.5-fold increase of delta2 and acetylated tubulin levels, which occurred at the onset of axonal degeneration. Furthermore, DRG axonal mitochondrial motility was decreased by BTZ. Finally, BTZ treated DRG neurons had no differential protein expression of OXPHOS subunits compared to untreated DRG neurons, whereas increased Mitofusin-2 protein expression and bioenergetics deficits were reported. In summary, our results provide support for the role of MT stability and mitochondrial dysfunction in the development of BTZ mediated neuropathy. Understanding these pathways may provide therapeutic targets for the treatment of this debilitating complication.

The authors note that on page 2, left column, first full paragraph, line 9, the citations to ref. 3 and ref. 41 are incorrect and thus should have been removed. The authors also note that Fig. 5 appeared incorrectly. In panel A, the one-letter symbol for glutamic acid appeared incorrectly as "G" and should instead have appeared as "E." The corrected figure and its legend appear below. The online version has been corrected.

Introduction: Chemotherapy-induced peripheral neurotoxicity (CIPN) is one of the dose-limiting side effects for several effective anticancer drugs. In case of severe neuropathy, chemotherapy dose reduction/withdrawal can occur, with possible negative effects on oncological prognosis. ncomplete knowledge of CIPN pathogenesis is the main reason why therapeutic strategies are not currently available. Herein we evaluated the onsequences of CIPN on oxidative stress and mitochondria dynamics using in vivo and in vitro models. Methods: Adult male Wistar rats were treated with chemotherapeutic agents (paclitaxel (PTX) 10 mg/kg 1 qw, i.v; oxaliplatin (OHP) 5mg/kg 2qw, i.v; 5 fluorouracil (5FU) 50 mg/kg 1 qw i.p, the latter used as non CIPN-promoting chemotherapeutic drug) or vehicle for 4 or 6 weeks and multimodal assessment of CIPN was performed (neurophysiology nd behavioral test). Oxidative stress, gene and protein expression were evaluated in dorsal root ganglia (DRG) of treated animals; moreover, in vitro nalyses of mitochondria motility were performed. Results: PTX-treated rats showed allodynia and neurophysiological alterations. Molecular analyses of RG isolated from PTX-treated rats indicated lack of oxidative stress; however, the expression of the key markers associated with mitochondrial dynamics was altered. Conversely, OHP- or 5FU-treated rats did not show similar mechanical allodynia, nor presented modifications in markers associated with mitochondrial dynamics. However, OHP selectively affected mitochondrial electron transport chain protein levels and induced oxidative stress. We measured mitochondria motility in cultured adult DRG neurons exposed to doses at which OHP induced axonal degeneration in vitro and found that OHP promoted mitochondrial motility prior to axonal degeneration. Conclusions: Altogether our data support the notion that allodynia specifically orrelates with perturbation of mitochondria dynamics and suggest that seemingly unrelated CIPN drug

Introduction: Bortezomib (BTZ) is a proteasome inhibitor used for the treatment of multiple myeloma. BTZ treatment results in axonal degeneration, producing a disabling painful toxic peripheral neuropathy in approximately 50% of patients. In the current study, we examined whether treatment with BTZ for 24 hours would affect microtubule(MTs) stability as well as axonal transport and oxidative phosphorylation in primary cultured dorsal root anglion (DRG) sensory neurons. Methods: MT stability was indirectly measured by western blotting and quantitative confocal microscopy of delta2- tubulin and tubulin acetylation, while axonal mitochondrial trafficking was evaluated by time-lapse confocal microscopy and kymograph analysis. To valuate the effects of BTZ on the generation and regulation of cellular bioenergetics, mitochondrial oxidative phosphorylation (OXPHOS) was assessed via western blot analysis of key molecular markers. Results: BTZ treatment of cultured DRG sensory neurons induced an approximately 2.5-fold increase of delta2 and acetylated tubulin levels, which occurred at the onset of axonal degeneration. Furthermore, DRG axonal mitochondrial motility and trafficking were decreased by BTZ; these changes occurred independently of the direction of mitochondrial trafficking. Finally, BTZ treated DRG neurons had differential protein expression of OXPHOS subunits compared to untreated DRG neurons. Conclusions: In summary, our results demonstrate that BTZ effects both MT stability and mitochondrial function in DRG neurons, supporting the idea that both loss of normal tubulin and mitochondria function together promote the development of BTZ mediated neuropathy. This work is supported by Fondazione Cariplo, Grant # 2019-1482

The pathogenesis of chemotherapy-induced peripheral neuropathy (CIPN) is poorly understood. Here, we report that the CIPN-causing drug bortezomib (Bort) promotes delta 2 tubulin (D2) accumulation while affecting microtubule stability and dynamics in sensory neurons in vitro and in vivo and that the accumulation of D2 is predominant in unmyelinated fibers and a hallmark of bortezomib-induced peripheral neuropathy (BIPN) in humans. Furthermore, while D2 overexpression was sufficient to cause axonopathy and inhibit mitochondria motility, reduction of D2 levels alleviated both axonal degeneration and the loss of mitochondria motility induced by Bort. Together, our data demonstrate that Bort, a compound structurally unrelated to tubulin poisons, affects the tubulin cytoskeleton in sensory neurons in vitro, in vivo, and in human tissue, indicating that the pathogenic mechanisms of seemingly unrelated CIPN drugs may converge on tubulin damage. The results reveal a previously unrecognized pathogenic role for D2 in BIPN that may occur through altered regulation of mitochondria motility.

Il contributo intende affrontare la prassi statutaria dei più recenti partiti politici, al fine di evidenziare la "crisi" della democrazia interna delle organizzazioni politiche. Ciò, almeno, sotto un duplica aspetto. Da un lato sono codificate regole che attribuiscono ampi poteri a pochi soggetti. Dall'altro, vi è la tendenza a superare le regole statutarie, privilegiando le scelte e ed i diktat di coloro che guidano il partito politico. Nella parte finale del contributo si dialoga con il futuro legislatore al fine di individuare alcune linee guida per una disciplina organica dei partiti politici.

Bortezomib (BTZ) is a proteasome inhibitor that represents the cornerstone in the treatment of multiple myeloma. Despite its efficacy, its clinical use is limited by severe painful peripheral neuropathy (BIPN) that occurs approximately in 50% of patients, impairing their life and representing a dose- imiting toxicity. There is increasing evidence that, instead of BTZ proteasome inhibition activity, BIPN may be due to BTZ off-targets. We focused our attention on tubulin and mitochondrial trafficking. In fact, on one hand, post-translational tubulin modification and Microtubule Associated Proteins MAPs) regulate microtubules (MTs) dynamics, fundamental for nervous system functions. On the other hand, axonal transport of mitochondria along the axons is essential for metabolic state and synaptic activity. Alterations of these two processes and modifications in their dynamics could lead to BIPN. In this work, we evaluated how these aspects take place in neurons isolated from adult mice dorsal root ganglia and treated with BTZ. MAP 2, delta2 tubulin a marker of hyperstable MTs) and tubulin acetylation were assessed through western blot analysis while the axonal mitochondrial trafficking as evaluated by time lapse confocal microscopy and kymograph analysis. We investigated the MTs stabilization by MAPs and by posttranslational modifications of tubulin and we found that BTZ treatment induced alterations of MAP2, delta2 tubulin and acetylated tubulin proteins quantity. After treatment for 24h with BTZ we also observed a loss of mitochondrial motility and dose-dependent reduction of transport speed, but no alterations were observed on the direction of the movement. Our results demonstrated that BTZ affect both mitochondrial trafficking and tubulin cytoskeleton in sensory neurons. Moreover, our data There is increasing evidence that, instead of BTZ proteasome inhibition activity, BIPN may be due to BTZ off-targets. We focused our attention on tubulin and mitochondrial traffick